Activated platelets secrete a host of soluble mediators that play critical roles in proper blood clot formation, inflammation, angiogenesis, and other physiological responses. In resting platelets, most of these mediators are stored within at least two distinct classes of secretory granules: protein mediators such as fibrinogen and von Willebrand factor are stored in 1 granules, whereas ADP, ATP, calcium and serotonin are stored in dense granules. These granules are generated within megakaryocytes and sequestered into forming platelets. Both types of granules are thought to share some features with lysosomes, but how they are formed and how their contents are specifically segregated from traditional lysosomal contents within megakaryocytes is not known. Defects in the formation of either class of granule, or in the storage of specific components within them, result in prolonged bleeding and abnormal platelet aggregation. In particular, dense granules and tissue-specific lysosome-related organelles in several other cell types, including melanosomes in pigment cells, are malformed in patients with Hermansky-Pudlak Syndrome (HPS), a group of disorders with mutations in any of at least eight genes. We have made considerable progress in understanding how the products of several HPS-associated genes function in trafficking of proteins from endosomes to melanosomes, and have characterized cytoplasmic sorting signals that interact with some of these gene products and mediate sorting of integral membrane proteins specifically to melanosomes. In this proposal, we will exploit our knowledge of the melanocyte, the availability of two unique megakaryocyte culture systems, and a recently discovered integral membrane protein on dense granules to test the hypothesis that similar pathways regulate protein sorting to melanosomes in melanocytes and to dense granules in megakaryocytes, and that these pathways are distinct from those that regulate protein sorting to 1 granules. We will also test the hypothesis that proper targeting of proteins specifically to dense granules requires HPS-associated gene products. Our Specific Aims are: 1. To test whether dense granule proteins localize to melanosomes in melanocytes and whether melanosomal proteins localize to dense granules in megakaryocytes. 2. To test whether cytoplasmic targeting signals like those on melanosomal proteins are necessary and/or sufficient to target SLC35D3 to dense granules or their precursors. 3. To test whether dense granule proteins are differentially mislocalized to endosomes in megakaryocytes that lack different HPS-associated gene products. PUBLIC HEALTH RELEVANCE: In order for blood clots to form during tissue damage, platelets must be activated to release a number of components from specialized granules called 1 and dense granules. Formation of these granules is a clinically important process because their malformation underlies a number of genetic and acquired bleeding disorders, but very little is known about how they form and accumulate their contents. In this project, we will exploit our understanding of the formation of a different but related organelle in pigment cells to determine how the platelet granules are formed and what goes wrong in certain heritable bleeding disorders.